A. Field Of the Invention
This invention is in the field of separation of biomolecules and, in particular, separations by capillary electrophoresis and the use of the capillary electrophoresis to detect such molecules.
B. Background of the Prior Art
Electrophoresis is a separation process in which molecules with a net charge migrate through a medium under the influence of an electric field. Traditionally, slab gel electrophoresis has been a widely used tool in the analysis of genetic materials. See, for example, G. L. Trainor, Anal. Chem., 62, 418-426 (1990). Capillary electrophoresis has emerged as a powerful separation technique, with applicability to a wide range of molecules from simple atomic ions to large DNA fragments. In particular, capillary electrophoresis has become an attractive alternative to slab gel electrophoresis for biomolecule analysis, including DNA sequencing. See, for example, Y. Baba et at., Trends in Anal. Chem., 11, 280-287 (1992). This is generally because the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, capillary electrophoresis requires less sample and produces faster and better separations than slab gels.
Currently, sophisticated experiments in chemistry and biology, particularly molecular biology, involve evaluating large numbers of samples. For example, DNA sequencing of genes is time consuming and labor intensive. In the mapping of the human genome, a researcher must be able to process a large number of samples on a daily basis. If capillary electrophoresis can be conducted and monitored simultaneously on many capillaries, i.e., multiplexed, the cost and labor for such projects can be significantly reduced. Attempts have been made to sequence DNA in slab gels with multiple lanes to achieve multiplexing. However, slab gels are not readily amenable to a high degree of multiplexing and automation. Difficulties exist in preparing uniform gels over a large area, maintaining gel to gel reproducibility and loading sample wells. Furthermore, difficulties arise as a result of the large physical size of the separation medium, the requirements for uniform cooling, large amounts of media, buffer, and samples, and long run times for extended reading of nucleotide sequences. Unless capillary electrophoresis can be highly multiplexed and multiple capillaries run in parallel, the advantages of capillary electrophoresis cannot produce substantial improvement in shortening the time needed for sequencing the human genome.
Capillary electrophoresis possesses several characteristics which makes it amenable to this application. The substantial reduction of Joule heating per lane makes the overall cooling and electrical requirements more manageable. The cost of materials per lane is reduced because of the smaller sample sizes. The reduced band dimensions are ideal for excitation by laser beams, as well as focused extended sources, and for imaging onto array detectors or discrete spot detectors. The concentration of analyte into such small bands results in high sensitivity. The use of electromigration injection, i.e., applying the sample to the capillary by an electrical field, provides reproducible sample introduction with little band spreading, minimal sample consumption, and little labor.
Among the techniques used for detecting target species in capillary electrophoresis, laser-excited fluorescence detection so far has provided the lowest detection limits. Therefore, fluorescence detection has been used for the detection of a variety of analytes, especially macromolecules, in capillary electrophoresis. For example, Zare et al. (U.S. Pat. No. 4,675,300) discusses a fluoroassay method for the detection of macromolecules such as genetic materials and proteins in capillary electrophoresis. Yeung et al. (U.S. Pat. No. 5,006,210) presented a system for capillary zone electrophoresis with laser-induced indirect fluorescence detection of macromolecules, including proteins, amino acids, and genetic materials.
Systems such as these generally involve only one capillary. There have been attempts to implement the analysis of more than one capillary simultaneously in the electrophoresis system, but the number of capillaries has been quite small. For example, S. Takahashi et al., Proceedings of Capillary Electrophoresis Symposium, December, 1992, referred to a multi-capillary electrophoresis system in which DNA fragment samples were analyzed by laser irradiation causing fluorescence. This method, however, relies on a relatively poor focus (large focal spot) to allow coupling to only a few capillaries. Thus, this method could not be applied to a large number of capillaries. This method also results in relatively low intensity and thus poor sensitivity because of the large beam. Furthermore, detection in one capillary can be influenced by light absorption in the adjacent capillaries, thus affecting accuracy due to cross-talk between adjacent capillaries.
Attempts have been made to perform parallel DNA sequencing runs in a set of up to 24 capillaries by providing laser-excited fluorometric detection and coupling a confocal illumination geometry to a single laser beam and a single photomultiplier tube. See, for example, X. C. Huang et at., Anal. Chem., 64, 967-972 (1992), and Anal. Chem., 64, 2149-2154 (1992). Also see U.S. Pat. No. 5,274,240. However, observation is done one capillary at a time and the capillary bundle is translated across the excitation/detection region at 20 mm/see by a mechanical stage; the capillaries in this system are not transportable to a different site for measurement.
There are features inherent in the con focal excitation scheme that limit its use for very large numbers of capillaries. Because data acquisition is sequential and not truly parallel, the ultimate sequencing speed is generally determined by the observation time needed per DNA band for an adequate signal-to-noise ratio. Moveover, the use of a translational stage can become problematic for a large capillary array. Because of the need for translational movement, the amount of cycling and therefore bending of the capillaries naturally increases with the number in the array. It has been shown that bending of the capillaries can result in loss in the separation efficiency. This is attributed to distortions in the gel and multipath effects. Sensitive laser-excited fluorescence detection also requires careful alignment both in excitation and in light collection to provide for efficient coupling with the small inside diameter of the capillary and discrimination of stray light. The translational movement of the capillaries thus has to maintain stability to the order of the confocal parameter (around 25 .mu.m) or else the cylindrical capillary walls will distort the spatially selected image due to misalignment of the capillaries in relation to the light source and photodetector. In addition, long capillaries provide slow separation, foul easily, and are difficult to replace.
U.S. Pat. No. 5,324,401 to Yeung et al. describes a multiplexer capillary electrophoresis system where excitation light is introduced through an optical fiber inserted into the capillary. In this system the capillaries remain in place, i.e. in the buffer solutions when the capillaries are read.
U.S. Pat. No. 5,332,480 (Dalton et al.) describes a multiple capillary electrophoresis device for continuous batch electrophoresis.
U.S. Pat. No. 5,277,780 (Kambara) describes a two dimensional capillary electrophoresis apparatus for use with a two dimensional array of capillaries for measuring samples, such as DNA samples, in an array of test wells.
U.S. Pat. No. 5,338,427 (Shartle et at.) describes a single use capillary cartridge having electrically conductive films as electrodes; the system does not provide for multiplexed sampling, sample handling, and electrophoresis.
U.S. Pat. Nos. 5,091,652 (Mathies et at.) and 4,675,300 (Zare et al.) describe means for detecting samples in a capillary.
U.S. Pat. No. 5,372,695 (Demorest) describes a system for delivering reagents to serve a fix capillary scanner system.
Numerous examples of sample handling for capillary electrophoresis are known. For example, James in U.S. Pat. No. 5,286,652 and Christianson in U.S. Pat. No. 5,171,531 are based on presenting a single vial of sample to a single separation capillary for a sequential series of analyses.
Goodale in U.S. Pat. No. 5,356,625 describes a device for presentation of a tray of 7 vials of sample to an array of seven capillaries for the sample injection process.
Carson in U.S. Pat. No. 5,120,414 describes injection of a sample contained within a porous membrane onto a single-capillary electrophoresis device. The end of the capillary must be in intimate contact with the porous membrane to affect sample introduction into the capillary.
In contrast, the present invention provides short disposable capillaries mounted in a frame which is integral with a liquid handling system. This system permits rapid multiplexed approach to capillary electrophoresis.
Numerous examples of multi-well devices with integral membranes are known (e.g. Mann in U.S. Pat. No. 5,043,215, Matthis in U.S. Pat. No. 4,927,604, Bowers in U.S. Pat. No. 5,108,704, Clark in U.S. Pat. No. 5,219,528). Many of these devices attach to a base unit which can be evacuated, drawing samples through the membrane for filtration.
Numerous examples of multi-channel metering devices such as multi-channel pipettes are known. One example is described in a device by Schramm in U.S. Pat. No. 4,925,629, which utilizes an eight-channel pipette to meter samples/reagents to/from multi-well plates. A second example is a 96-channel pipetting device described by Lyman in U.S. Pat. No. 4,626,509. These devices use positive displacement plungers in corresponding cylinders to draw in and expel liquid in the sampling/metering step.
Finally, Flesher in U.S. Pat. No. 5,213,766 describes a 96-channel device which contains flexible "fingers" which can be deformed out of a common plane; each "finger" can be deflected into a well of a multi-well plate to acquire a small aliquot of sample by one of several mechanisms.
The present invention differs in that it provides for simultaneously sampling of an array as samples; simultaneously handling the samples and presenting an array of the samples for capillary electrophoresis; simultaneously transferring the array of presented samples to an array of capillaries; and simultaneously conducting separations in the capillary electrophoresis columns.